Process for the production of biologically active peptide via the expression of modified storage seed protein genes in transgenic plants

Abstract

A process is described for producing a polypeptide by cultivating a plant whose genome contains a recombinant DNA. The recombinant DNA includes: a) a first DNA sequence which encodes a precursor of a 2S albumin; and b) a heterologous second DNA sequence that encodes the polypeptide and that is inserted into, or replaces at least in part, a hypervariable region of the first DNA between codons which code for fourth and fifth cysteine residues of the large subunit of the 2S albumin. The ends of the second DNA sequence are each linked to one or more codons that encode one or more amino acid residues, to define first selectively cleavable border sites surrounding the polypeptide for separating the polypeptide from surrounding parts of the 2S albumin; and the hypervariable region, containing the second DNA sequence, encodes no more than approximately the same number of amino acids as are encoded by the hypervariable region without the second DNA sequence.

We claim: 
     
       1. A process for producing a polypeptide, which process comprises the steps of: (a) cultivating a seed-forming plant, said plant comprising in its genome a chimeric DNA construct encoding a precursor of a modified 2S albumin wherein said chimeric DNA construct comprises: (i) a first DNA sequence encoding a precursor of a 2S albumin which is modified in the hypervariable region encoding the region between a fourth cysteine residue and a fifth cysteine residue of a large subunit of said 2S albumin by replacing all or part of said hypervariable region with a heterologous second DNA sequence encoding said polypeptide, wherein the ends of said second DNA sequence are linked to one or more codons encoding one or more amino acid residues to define first selectively clearable border sites; and   (ii) a seed specific promoter; wherein said modified 2S albumin is expressed and accumulated in the seeds of said plant;     (b) isolating from the seeds of said plant said modified 2S albumin;   (c) cleaving said modified 2S albumin at said cleavable border sites; and   (d) recovering said polypeptide.   
     
     
       2. The process of claim 1, wherein said polypeptide is formed of repeats of units of a biologically active polypeptide or protein and said units are separated from one another by means of second selectively cleavable border sites. 
     
     
       3. The process of claim 1, wherein said seed-specific promoter is heterologous to said first DNA sequence. 
     
     
       4. The process of claim 1, wherein said polypeptide is a labeled protein comprising atoms selected from the group consisting of carbon 14, nitrogen 15, hydrogen 3, sulphur 35 and phosphorous 32. 
     
     
       5. The process of claim 1, wherein said first DNA sequence is naturally associated with said plant. 
     
     
       6. The process of claim 1, wherein said first DNA sequence is heterologous to said plant. 
     
     
       7. The process of claim 1, wherein said seed-specific promoter is heterologous to said seed-forming plant. 
     
     
       8. The process of claim 1, wherein said seed-specific promoter is naturally associated with said seed-forming plant. 
     
     
       9. The process of claim 1, wherein said hypervariable region in said chimeric DNA construct starts three codons downstream of said codon encoding said fourth cysteine residue and ends three codons upstream of said codon encoding said fifth cysteine residue. 
     
     
       10. The process of claim 1, wherein said hypervariable region starts six codons downstream of said codon encoding said fourth cysteine residue and ends six codons upstream of said codon encoding said fifth cysteine residue. 
     
     
       11. The process of claim 1, wherein said second DNA sequence is located between codons which code for amino acids 31 and 57 of the large subunit of a 2S albumin of Arabidopsis thaliana. 
     
     
       12. The process of claim 1, wherein said 2S albumin is selected from the group consisting of 2S albumins of Ricinus communis, Arabidopsis thaliana, Brassica napus and Bertholletia excelsa. 
     
     
       13. The chimeric DNA construct of claim 1. 
     
     
       14. The chimeric DNA construct of claim 9. 
     
     
       15. The chimeric DNA construct of claim 10. 
     
     
       16. The chimeric DNA construct of claim 11. 
     
     
       17. The chimeric DNA construct of claim 12. 
     
     
       18. A plant containing the chimeric DNA construct of claim 13 and its seeds. 
     
     
       19. A plant containing the chimeric DNA construct of claim 14 and its seeds. 
     
     
       20. A plant containing the chimeric DNA construct of claim 15 and its seeds. 
     
     
       21. A plant containing the chimeric DNA construct of claim 16 and its seeds. 
     
     
       22. A plant containing the chimeric DNA construct of claim 17 and its seeds.